FDA-approved cannabidiol [Epidiolex®] alleviates Gulf War Illness-linked cognitive and mood dysfunction, hyperalgesia, neuroinflammatory signaling, and declined neurogenesis

Background Chronic Gulf War Illness (GWI) is characterized by cognitive and mood impairments, as well as persistent neuroinflammation and oxidative stress. This study aimed to investigate the efficacy of Epidiolex®, a Food and Drug Administration (FDA)-approved cannabidiol (CBD), in improving brain function in a rat model of chronic GWI. Methods Six months after exposure to low doses of GWI-related chemicals [pyridostigmine bromide, N,N-diethyl-meta-toluamide (DEET), and permethrin (PER)] along with moderate stress, rats with chronic GWI were administered either vehicle (VEH) or CBD (20 mg/kg, oral) for 16 weeks. Neurobehavioral tests were conducted on 11 weeks after treatment initiation to evaluate the performance of rats in tasks related to associative recognition memory, object location memory, pattern separation, and sucrose preference. The effect of CBD on hyperalgesia was also examined. The brain tissues were processed for immunohistochemical and molecular studies following behavioral tests. Results GWI rats treated with VEH exhibited impairments in all cognitive tasks and anhedonia, whereas CBD-treated GWI rats showed improvements in all cognitive tasks and no anhedonia. Additionally, CBD treatment alleviated hyperalgesia in GWI rats. Analysis of hippocampal tissues from VEH-treated rats revealed astrocyte hypertrophy and increased percentages of activated microglia presenting NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) complexes as well as elevated levels of proteins involved in NLRP3 inflammasome activation and Janus kinase/signal transducers and activators of the transcription (JAK/STAT) signaling. Furthermore, there were increased concentrations of proinflammatory and oxidative stress markers along with decreased neurogenesis. In contrast, the hippocampus from CBD-treated GWI rats displayed reduced levels of proteins mediating the activation of NLRP3 inflammasomes and JAK/STAT signaling, normalized concentrations of proinflammatory cytokines and oxidative stress markers, and improved neurogenesis. Notably, CBD treatment did not alter the concentration of endogenous cannabinoid anandamide in the hippocampus. Conclusions The use of an FDA-approved CBD (Epidiolex®) has been shown to effectively alleviate cognitive and mood impairments as well as hyperalgesia associated with chronic GWI. Importantly, the improvements observed in rats with chronic GWI in this study were attributed to the ability of CBD to significantly suppress signaling pathways that perpetuate chronic neuroinflammation. Supplementary Information The online version contains supplementary material available at 10.1186/s40779-024-00563-2.

10).Sixteen weeks after the restraint stress regimen, animals in both groups were interrogated with a series of neurobehavioral tests.After completion of neurocognitive tests, these rats were deeply anesthetized with isoflurane, and their brains were fixed with 4% paraformaldehyde via intracardiac perfusions.Fixed brain tissues were processed for immunohistochemical and immunofluorescence studies.The Institutional Animal Care and Use Committee of Texas A&M University approved all studies conducted in this investigation (Animal Use Protocol number: 2022-0166D).

Fifteen-minute of restraint stress daily for 28 d in naï ve rats does not lead to object recognition memory impairment
To evaluate a recognition memory task that depends on the function of the hippocampus and the perirhinal cortex, each animal underwent a novel object recognition test (NORT), as detailed in our previous studies [1,2] (Additional file 1: Fig. S1a).Animals proficient in object recognition memory tend to explore a novel object (NO) over a familiar object (FO).In trial 3 (performed 30 min after trial 2), naï ve control rats preferred the NO vs. the FO (P < 0.0001, Additional file 1: Fig. S1b).Remarkably, naï ve control rats subjected to 15-minute restraint stress daily for 28 d also maintained proficiency for object recognition memory by exploring the NO for prolonged periods than the FO (P < 0.05, Additional file 1: Fig. S1b).
Analysis of the discrimination index (DI) for the NO revealed no differences in object recognition memory between the age-matched naï ve group and the group subjected to the stress (P > 0.05, Additional file 1: Fig. S1c).Thus, 15 min of restraint stress daily for 28 d in naï ve rats does not impair object recognition memory.

Fifteen-minute of restraint stress daily for 28 d in naï ve rats does not lead to pattern separation impairment
We next evaluated the pattern separation function, which depends upon the integrity of the dentate gyrus in the hippocampus, including the extent of neurogenesis [3,4].The test involved 4 trials with an intertrial interval of 30 min.The test was performed as described in our previous studies [2].In pattern separation test (PST), animals proficient in pattern separation spend more time exploring the NO on pattern 2 (NO on P2) than the FO on pattern 2 (FO on P2) in T4 (Additional file 1: Fig. S2a).The ability for pattern separation was observed in both naï ve rats and naï ve rats that underwent 15 min of restraint stress daily for 28 d, as they displayed a higher affinity to explore the NO on P2 vs. the FO on P2 (P < 0.05, Additional file 1: Fig. S2b).Analysis of the NO on P2 -DI confirmed that pattern separation function did not differ between naï ve rats and naï ve rats that underwent stress (P > 0.05, Additional file 1: Fig. S2c).Thus, 15 min of restraint stress daily for 28 d in naï ve rats does not impair pattern separation function.

Fifteen-minute of restraint stress daily for 28 d in naï ve rats does not lead to anhedonia
The anhedonia was assessed through a sucrose preference test (SPT), as described in our previous reports [2,5,6].In SPT, naï ve rats and naï ve rats subjected to stress (15 min of restraint stress daily for 28 d) preferred sucrose water over standard water, implying no anhedonia (P < 0.01, Additional file 1: Fig. S3a).Furthermore, the sucrose preference rate (SPR) of naï ve rats and naï ve rats subjected to stress were comparable (P > 0.05, Additional file 1: Fig. S3b).Also, the total fluid consumption did not differ between the two groups (P > 0.05, Additional file 1: Fig. S3b).Thus, 15 min of restraint stress daily for 28 d in naï ve rats does not result in anhedonia.

Fifteen-minute of restraint stress daily for 28 d in naï ve rats does not activate microglia in the hippocampus
We analyzed the possible chronic activation of microglia in the hippocampus of naï ve rats subjected to stress via visualization of IBA-1 + microglia expressing CD68 (a marker of activated microglia) through dual immunofluorescence for IBA-1 and CD68 (Additional file 1: Fig. S4).Analysis of the percentage of IBA-1 + cells expressing CD68 revealed similar percentages of microglia displaying CD68 between naï ve rats and naï ve rats that underwent stress (P > 0.05, Additional file 1: Fig. S4).Thus, 15 min of restraint stress daily for 28 d in naï ve rats does not lead to chronic activation of microglia in the hippocampus.

Fifteen-minute of restraint stress daily for 28 d in naï ve rats does not alter neurogenesis in the hippocampus
Next, we quantified the status of hippocampal neurogenesis via stereological quantification of doublecortin-positive (DCX + ) newly born neurons in the subgranular zonegranule cell layer (SGZ-GCL).
The morphology and distribution of DCX + neurons in the SGZ-GCL from both groups are illustrated (Additional file 1: Fig. S5).The number of DCX + newly born neurons in the SGZ-GCL did not differ between the naï ve group and the naï ve group subjected to stress (P > 0.05, Additional file 1: Fig. S5).
Thus, 15 min of restraint stress daily for 28 d in naï ve rats does not lead to decreased neurogenesis in the hippocampus.

Cannabidiol (CBD) treatment did not cause weight loss in rats with chronic GWI
Since one of the adverse effects of CBD is decreased appetite, we monitored weekly body weights over 16 weeks in GWI rats that received VEH or CBD during the treatment regimen using repeated measures of two-way ANOVA.Interestingly, we did not observe statistically significant differences in body weights between the two groups during the 16-week treatment period (Additional file 1: Fig. S6).The examples of IBA-1 + microglia (green) expressing CD68 (red) in the hippocampal dentate gyrus from a naï ve rat and a naï ve rat that underwent stress (scale bar = 10 μm).Arrows denote IBA-1 + microglia expressing CD68.The bar chart compares percentages of IBA-1 + microglia expressing CD68 in the hippocampus between the two groups.Please refer to Table S4 in Additional file 1 for detailed statistical information.IBA-1 ionized calcium-binding adapter molecule-1, CD68 cluster of differentiation 68

Fig. S1
Fig. S1 Fifteen-minute of restraint stress daily for 28 d in naï ve rats does not impair object recognition memory.a The sequence of trials in a NORT.b Percentages of the TOETs spent with the FO and the NO in naï ve rats and naï ve rats subjected to stress (n = 10).c The NO DI between the two groups.* P < 0.05, **** P < 0.0001.Please refer to Table S4 in Additional file 1 for detailed statistical information.NORT novel object recognition test, T trial, FO familiar object, NO novel object, TOET total object exploration times, DI discrimination index

Fig. S2
Fig. S2 Fifteen-minute of restraint stress daily for 28 d in naï ve rats does not impair their pattern separation ability.a The sequence of trials in a PST.b Percentages of the TOETs spent with the FO on P2 and the NO on P2 in naï ve rats and naï ve rats subjected to stress (n= 10).c The NO on the P2 DI between the two groups.* P < 0.05, ** P < 0.01.Please refer to Table S4 in Additional file 1 for detailed statistical information.PST pattern separation test, T trial, FO on P2 familiar object on pattern 2, NO on P2 novel object on pattern 2, TOET total object exploration time, DI discrimination index

Fig. S3
Fig. S3 Fifteen-minute of restraint stress daily for 28 d in naï ve rats does not cause anhedonia.a The volumes of standard water and sucrose-containing water consumed by animals in naï ve rats and naï ve rats subjected to stress (n = 10) in the SPT.b SPR and total volume consumed between the two groups.** P < 0.01.Please refer to Table S4 in Additional file 1 for detailed statistical information.SPR sucrose preference rate, SPT sucrose preference test

Fig. S4
Fig. S4 Fifteen-minute of restraint stress daily for 28 d in naï ve rats does not cause microglia activation.

Fig. S5
Fig. S5 Fifteen-minute of restraint stress daily for 28 d in naï ve rats does not cause changes in hippocampal neurogenesis.Figures illustrate examples of DCX + newly born neurons from a naï ve rat and a naï ve rat subjected to stress (scale bar = 25 µm).The bar chart compares the number of DCX + newborn neurons between naï ve rats and the naï ve rats subjected to stress.Please refer to Table S4 in Additional file 1 for detailed statistical information.ML molecular level, GCL granule cell layer, SGZ subgranular zone, DCX doublecortin